Core 2 will be responsible for the growth and genetic modification of fibroblasts that will be used for intracerebral grafting as described in projects 1, 2 and 3. In addition to service components, the core project will explore methods for optimizing the growth of primary fibroblasts in vitro and maximizing gene expression in these cells. Fibroblasts obtained from skin biopsies will be grown on modified plastic substratum (e.g. fibronectin) or in the presence of growth factors (e.g. epidermal growth factor) in order to optimize the growth and survival of fibroblasts in culture, particularly after genetic modification, while avoiding cell transformation. In order to achieve maximal and stable gene expression in primary fibroblasts, particularly while they are in a quiescent state, the activity of different viral and non-viral promoters will be examined. The possibility of increasing gene expression by inserting enhancer regions will be explored. Certain promoters (e.g. collagen type I promoters) can be regulated by cytokines or growth factors. Determining the parameters for obtaining increased gene expression from regulatable promoters will also be examined. However, the final measure of the optimal activity of promoter(s) is in vivo and adequate gene expression in vitro may or may not predict gene expression in vivo. This research will enable us to find promoter(s) with optimal activity in primary rat fibroblasts in vitro and in vivo which may be regulated by external agents. The use of an optimal promoter, coupled with the ability to enhance fibroblast growth, will allow us to generate genetically modified fibroblasts best suited for implantation.